Syntrichia caninervis adapt to mercury stress by altering submicrostructure and physiological properties in the Gurbantünggüt Desert

Sewage and industrial waste discharges have been found to have a deleterious effect on plant growth and environmental safety through the accumulation of trace metal mercury (Hg) in soils. Although the effects of Hg on vascular plants have been reported in terms of enzyme activity, oxidative damage and physiology, few studies have been done on non-vascular plants. A simulation experiment including 7 Hg concentrations (0, 10, 20, 30, 40, 50, 75 µM) was conducted to investigate the influence of Hg stress on ultrastructure and physiological properties of biocrust moss Syntrichia. caninervis across 7 consecutive days. The results showed that the lowest lethal concentration of S. caninervis was 30 µM Hg. The mortality rate of the plants increased significantly with Hg concentrations. The ultrastructure did not change significantly at Hg concentration ≤ 20 µM, while exceeding which, cell walls began to separate, nuclei began to blur, and chloroplasts began to expand. The soluble sugars (SS), peroxidase (POD), and superoxide dismutase (SOD) activities increased initially and then decreased with the increase of concentration in the time gradient, with the largest values at 20 µM. The contents of malondialdehyde (MDA) and proline (Pro) increased with the increase of Hg concentration, both reached peak value at 50 µM. However, chlorophyll (Chl) contents continued to decrease along both the concentration and time gradients. Pearson correlation and principal component analysis showed that two principal components (PC1 and PC2) explained 73.9% of the variance in plant adaptation to Hg stress. SOD, POD, Chl, SS, and Pro all responded well to Hg in S. caninervis. Our study showed that Hg stress caused changes in ultrastructure and physiological metabolism of S. caninervis. 20 µM was the maximum concentration of Hg that biocrust moss S. caninervis can tolerate. S. caninervis mainly adopted two adaptation strategies related to exclusion and accumulation to reduce Hg stress.

Plant culture and treatment. S. caninervis were sampled in May 2019 by using a small shovel at the southern margin of the Gurbantünggüt Desert. More specifically, the S. caninervis with good growth and relatively uniform were firstly selected as sampling objects. Then, the surface sand was gently removed with a brush, and the samples were collected in petri dishes. After that, they were taken to the laboratory and cultured with deionized water for 2 days in an incubator at 25 ± 1 °C and 20% relative humidity (RH) 28 . After culturing for 2 days, S. caninervis individuals with about 0.8 cm in height were selected for further experiment using forceps, all samples (each sample includes 75 individuals) were placed in numbered petri dishes (d = 15 cm, V = 512.21 cm 3 ). They were treated with 0, 10,20,30,40,50, 75 µM Hg obtained from HgCl 2 water (DI) solution, and all roots should be immersed in solution. Finally, all samples were placed back to the incubator. All samples were cultured for 7 days, and the growth conditions of S. caninervis were observed on every day. During the experimental period (7 days), HgCl 2 solutions with each respective concentration were added into the petri dishes www.nature.com/scientificreports/ each day in order to maintain the Hg stress. Each Hg addition treatment was replicated three times with a total of 21 samples.
Determination of Hg contents. S. caninervi with different treatments of Hg after 7 days were collected and washed. Subsequently, they were air dried and grounded into powder with a mortar and pestle, 0.2 g dry samples were was taken in digestion tubes and 10 mL of HNO 3 , HClO 4 and H 2 SO 4 (5:1:1) were added to each tube and were kept for 12 h. The tubes were then placed at 80 °C in the digestion block for about 1 h and then raised the temperature slowly to 120-130 °C. When digestion was completed, the solutions were cooled, filtered and diluted to 100 mL with double deionized water. In the filtrates, the Hg was assayed using an atomic absorption spectrometer (Analyst 700 of Perkin Elmer) 29 .
Transmission electron microscopy (TEM). According to Khan et al. 30  Determination of chlorophyll contents. Chlorophyll a and b contents were determined by the method of Arnon 31 . Briefly, samples (0.3 g) were homogenized in acetone under dark conditions. Homogenates were centrifuged at 2800 g for 5 min, and the absorbance of the supernatant was measured at 645 and 663 nm. Then, the chlorophyll a and b contents and the total chlorophyll were determined. The total chlorophyll concentration was calculated utilizing the following formula: (12.7A 663 -2.69A 645 + 22.9A 645 -4.68A 663 ).
Determination of proline and soluble sugar. Proline (Pro) content was measured according to the method of Bates et al. 32 . A quantity (0.5 g) of fresh aboveground material of S. caninervis was homogenized with 3% sulfosalicylic acid (5 mL) on ice. The homogenate was heated in a water bath at 100 °C for 15 min and then cooled to indoor temperature. The extract was centrifuged for 10 min at 4000 g. The supernatant (2 mL) was mixed with the same volumes of glacial acetic acid (2 mL) and acid ninhydrin (2 mL), and then held the mixed liquids in a bath at 100 °C for 1 h. The reaction mixture was extracted with toluene (4 mL) and absorbance was measured at 520 nm using a spectrophotometer. The Pro content was calculated from the standard curve. Soluble sugar (SS) content was measured by anthrone test 33 . Fresh samples (0.5 g) was extracted using 5 mL of distilled water in a boiling water bath for 10 min, and then centrifuged it at 4000g for 10 min. The reaction included 0.05 mL of supernatant, 0.95 mL of ethanol (80%, v/v), and 5 mL of anthrone (0.1%, v/v) and was incubated in a boiling water bath for 10 min. After cooling, the absorbance of solution was measured at 625 nm.
Determination of malondialdehyde contents. Malondialdehyde (MDA) contents were measured by thiobarbituric acid based on the colorimetric method described by Choudhury and Panda 34 . The reaction mixture consisted of 1 mL enzyme solution, 1 mL 50 mM phosphate buffers (pH 7.0) and 2 mL 10% trichloroacetic acid was heated at 100 °C for 15 min and then centrifuged at 4000g for 10 min. The absorbance of the supernatant was measured at 450 nm, 532 nm and 600 nm by a using spectrophotometer.
Assay of antioxidant enzymes. The superoxide dismutase (SOD) and peroxidase (POD) activities were measured based on the methods of Kumar et al. 35 and Pasquariello et al. 36 , respectively. Briefly, 0.5 g fresh sample was ground in an ice-cold mortar with enzyme extraction buffers (2 mL). The mixture was later centrifuged at 12,000g for 20 min at 4 °C. The supernatant was used for the measurement of POD and SOD activities. The SOD activity was determined by measuring the ability of the enzyme to inhibit the photochemical reduction of nitroblue tetrazolium. The absorbance was set to 560 nm and test value was recorded using a spectrophotometer. For the measurement of POD activity, the methylcatechol reaction mixture was added to the supernatant while the absorbance was set at 470 nm.

Statistical analysis.
All experimental data were presented as the mean ± SD. One-way ANOVA was used to test differences in physiological properties among the different concentrations of Hg addition. Differences between two groups were measured using Duncan test. The significance of differences was assessed at the 5% probability level (p < 0.05). Correlation analysis and principal component analysis (PCA) were performed to test Permission statement. This is a permission from Xinjiang University to collect samples of well development Syntrichia caninervis from the southern margin of the Gurbantünggüt Desert, were complied with relevant institutional, nation, and international guidelines and legislation and transferring them safely to physicochemical analysis and ultrastructure determination to Xinjiang University, College of Ecology and Environment. Mamtimin Sulayman formally identified the Syntrichia caninervis used in the study. The voucher specimen of the Syntrichia caninervis is not deposited in the public herbarium.

Results
Effects of different Hg stresses on the S. caninervis growth condition. The effect of Hg on S.
caninervis growth condition was evaluated by color changes. With the increase in the concentrations of Hg addition, S. caninervis would lose their green color and then became yellowish or black-brown due to greater growth retardation. Black-brown indicated that the individuals were dead. According to the observed survival of S. caninervis along the experimental period (7 days) ( Table 1), our results showed that the most S. caninervis individuals were green or yellow-green (no death was observed) while the concentrations Hg was less than 30 µM. In contrast, when Hg concentration was equal to or higher than 30 µM, the black-brown death individuals appeared after 5 days. As the increased in Hg concentration, the mortality rate of S. caninervis individuals increased continuously. Even on the 7th day, all plants died in 75 µM solution (Table 1).
Hg accumulation. The content of Hg in tissues of S. caninervis increased concurrently with the increase of external Hg concentration ( Effects of different Hg stresses on the ultrastructure. TEM analysis illustrated that the Hg stress changed the ultrastructure of S. caninervis, but the change degree depended on concentrations. The ultrastructure was compared between the control (0 µM) and low (20 µM), medium (30 µM) and high (50 µM) concentrations. More specifically, there are no structural alterations in the control group (0 µM) (Fig. 1A). The well-developed cell wall (CW), cell membrane (CM), cell nucleus and chloroplast (Chl) can be clearly seen in TEM images. The thylakoids were arranged regularly and tightly in the grana. Chloroplast has an obvious appearance and clear oval shape. Starch grains (SG) with small diameters were rarely observed in cells. In contrast, the ultrastructure changed obviously, and the cell shape became irregular in 20, 30 and 50 µM of Hg concentrations. However, the degree of ultrastructural change was also closely related to the concentration. Briefly, under the stress of 20 µM   www.nature.com/scientificreports/ Hg concentration, chloroplast morphology was good, but the cell membrane was slightly damaged, the nucleus (CN) was slightly deformed, the numbers of starch grains were decreased, and the nucleoli was still mounted in cell (Fig. 1B). However, after increasing Hg concentration larger than 30 µM, compared with the control and 20 µM groups, the thylakoid shape was swollen, and the thylakoid lamellae were loose and arranged randomly. Also, the unstable cell wall (CW) with scattered cell membrane (CM), the invisible nucleus (CN), nucleolus and nuclear membrane (NM), as well as the absence of starch grains were observed from TEM images (Fig. 1C,D).

Effects of different Hg stresses on the physiological properties.
Our results suggested that Hg stress have the significantly influences on physiological properties of S. caninervis. The chlorophyll (including chlorophyll a and chlorophyll b) contents decreased continuously with the increase in Hg concentration in all experimental period (from 1 to 7 days), whereas the responses of other properties were differed. More specifically, Pro content increased with Hg concentration in all experimental periods (Fig. 2C). The responses of SS,

Response of ultrastructure in the mesophyll cells to Hg stress.
Many studies have showed that plant organelles would undergo a series of morphological and structural changes to adapt to the damage and stress of the trace metal elements [37][38][39] . For example, Sergio et al. reported that trace metal elements cause the plasmolysis and an obvious thickening of cell wall due to the harmful influences on the intracellular structure of bryophytes 40 . Vecchia et al. found that most of the damage caused by trace metal elements was related to bio-  www.nature.com/scientificreports/ logical membrane, resulting in that the organelles with abundance in membrane structure are more vulnerable to serious damage than other structures 25 . Among many membrane-abundance organelles, chloroplasts and mitochondria may be the most severely damaged organelles by trace metal elements, because the latter hindered the division and expansion of these two organelles, as well as affected their shape and arrangement of the cystoid system 41,42 . In this study, we have obtained the similar results to the above studies. Previous studies have demonstrated that trance metal toxicity to plants is positively correlated to trance metals concentration in plant tissues 43,44 . Our results found that Hg accumulation in S. caninervis increased with the addition of Hg and had the negative impacts on its growth and cell ultrastructure. The mortality rate increased continuously with the increase of Hg concentration. Under the stress and toxicity of Hg, the chloroplast, thylakoid, cell walls and cell membranes were are damaged to varying degrees, as well as the plasmolysis were occurred. Also, the damage of Hg to ultrastructure of the mesophyll cells increased with its concentration. More specifically, there were no significant change in mesophyll cells and osmiophilic granules in S. caninervis at Hg concentrations ≤ 20 µM. All S. caninervis individuals were alive and most of them showed green in color. Starch granules were the major form of energy storage. These results suggested that Hg concentrations below 20 µM were not sufficient to produce harmful influence on BSCs. After that, when the Hg concentration increased to 20 µM, intracellular starch granules disappeared, which maybe be related to the resistance of biological crust to stress and toxicity caused by trace metal elements. The decrease of starch particles reflected the increase of energy consumption, which was used to improve the tolerance of the crust to metal toxicity. Under the stress of 30 µM Hg, the mesophyll cells of S. caninervis gradually showed the blurred cell walls, slight division and some deformation of cytoplasm, obvious swelling and vacuolization of chloroplasts, the blurred granules and stroma lamellae, and disintegration of surrounding organelles. All these indicated that chloroplast organelles began to be damaged and became more and more serious over time. This was also reflected in the appearance of plant. S. caninervis showed a black-brown color, indicating they died on days 6 of the experiment. More seriously, when Hg concentration was larger than 50 µM, the concentration of Hg on the cell wall exceeded the threshold that the cells can endure, leading to the serious vacuolation formation, even chloroplast disintegration, and the fuzzy and loss of cell nuclear membrane structure, and finally resulting in cytoplasm overflow and loss of thylakoid, stroma and lamellar structure. This finding was consistent with previous studies on changes in the ultrastructure (such as cell wall, nucleus and chloroplast) regarding the stress and toxicity of trace metal elements 45 .
In this study, we found that the number of osmiophilic granules in mesophyll cells increased first and then decreased with the increase of Hg concentration. This may be because the increase of osmiophilic granules are beneficials to providing lipid for membrane renewal and further maintaining the structural integrity of the membrane at low stress of trace metal element or in the first half of our experimental period. At the same time, osmophilic granules were degraded into sugars under the environmental stress, which in turn increased the sugar concentration in cells and improved the resistance of S. caninervis. However, the excessive concentrations of Hg that may surpass the tolerance limit of S. caninervis, would result in individual death. The shutoff or the decrease of S. caninervis metabolism caused the reduction of the number of osmophilic granules. Additionally, our results also showed that Hg stress significantly reduced chlorophyll content (chlorophyll a and b, as well as total chlorophyll content) ( Fig. 2A). This may be due to the fact that Hg stress caused the disintegration of chloroplast thylakoid, and as a result inhibited chlorophyll synthesis 46,47 . Response of physiological properties on Hg stress. Soluble sugar and free proline are key osmotic adjustment substances and a major indicator for plant tolerance to stress 48 . The changes of osmotic adjustment substances were conducive to maintaining the stability of cellular structure and cell osmotic pressure, particularly under severe or prolonged stress 49 . In this study, SS content increased firstly and then decreased with the increase of Hg stress. Low Hg concentration (≤ 30 µM) increased SS content, while high Hg concentration reduced it. This was probably because the increase of SS content can effectively reduce the water potential of cells and prevent dehydration caused by Hg stress in the early stage of experiment or under low concentration. In addition, starch granules can be converted into SS under the metal stress, which then increased the SS concentration in cell solution, thus enhancing the plant tolerance to metal stress. However, the exorbitant Hg stress (≥ 50 µM) may inhibit anabolism and growth of S. caninervis, then leading to the decrease of SS content. In addition to osmotic adjustment, Pro can also serve as molecular chaperone to stabilize protein structure and play a regulatory role in antioxidant system 50,51 . Under the condition of high metals stress, higher Pro content can maintain appropriate cell expansion pressure and protect cell structure from trace metal element damage.
In a normal growth environment, the production and removal of reactive oxygen species (ROS) are dynamic balance in plants. However, ROS balance would be disrupted if plants are stressed by drought, salinity, cold or trace metal elements. The overproduction of ROS will accumulate in plants, resulting in oxidative damage 52,53 . Antioxidant enzymes, such as POD and SOD can convert toxic superoxide radicals into harmless ions and also remove hydrogen peroxide in plants 54 . Therefore, it is considered to be an important substance for limiting ROS and improving plant tolerance to environmental stress. In our results, SOD and POD activities increased firstly and then decreased with the increase of the Hg addition concentration (Fig. 3E,F). This may indicate that the activation of ROS scavenging system was activated at the early stage of Hg stress. S. caninervis increased SOD and POD activities for reducing the damage of the superfluous ROS caused by the Hg stress, and enhancing the body's tolerance to Hg stress. However, the antioxidant enzyme system would be inhibited gradually with the increase of Hg stress, leading to the limitation to the scavenging ability of ROS, and then as a result decreasing SOD and POD activities due to the damages of the functional membrane and enzyme system of cells 55 . Meanwhile, as the increase of Hg stress, oxidative stress would be deteriorated and the excessive ROS could not be dissipated timely, resulting in the damage to the cytoplasmic membrane and the disruption of antioxidant enzyme system. www.nature.com/scientificreports/ As an important marker of lipid peroxidation, MDA content would be increased with the reduction of the oxidative cellular defense 56 . The increase of MDA is beneficial to reduce the damage of the accumulation of toxic ROS such as OH − and H 2 O 2 caused by environmental stress to lipids, proteins and nucleic acids, thus rapid the death of plants 57 . In this study, MDA content increased with the increase of Hg stress, and the change range was higher in the later stage of the experiment than in the earlier stage. These indicated that Hg concentration has a higher induction effect on the increase of MDA content. The slow increase of MDA content in 2-4 days may be due to the fact that the activation of resistance mechanisms in plants to delay ROS damage to cells. After 5-6 days, the long-term Hg stress intensified membrane lipid peroxidation due to the inhibition of plant defense enzyme system, resulting in the rapid increase of MDA content.
The adaptive strategies of S. caninervis to Hg stress. Plants are continuously faced with abiotic stress and have evolved various mechanisms or adaptive strategies to overcome them 58 . In this study, two main adaptive strategies of S. caninervis were found based on principal component analysis and Pearson correlation analyses to resist Hg stress. The first strategy is that by regulating osmotic pressure and photosynthesis. Chl and SS showed an overall decreasing trend with increasing Hg concentration. The decrease in Chl content indicates that S. caninervis has been subjected to the toxic effects of Hg, which inhibited photosynthesis. The increasing of SS content at Hg concentration < 30 µM improves the cytosol concentration, reduces the cellular water potential and enhances the cell water retention capacity 59 ; The significant decrease in SS content under high Hg stress (> 30 µM) indicates that Hg disrupts the photosynthetic system of S. caninervis, causing the breakdown of soluble sugars and inhibiting their transformation. Proline plays an important role in the protection of cell membrane structures, the stability of biomolecular structures and the decreasing of cellular water potential 60,61 . In our study, the sustained increase in Pro content suggests that S. caninervis may reduce cellular water potential, enhance cellular water retention capacity and stabilize biomolecular structure to adapt to Hg stress by increasing proline. The other adaptive strategy is via enhancing the elimination of free radicals to mitigate the damage of trace metals through the antioxidant enzyme system (including POD and SOD). At Hg concentration < 20 µM, the increases of POD and SOD can convert more toxic superoxide radicals into harmless ions in plant and remove hydrogen peroxide caused by trace metal stress at the same time 62 . This strategy is to enhance the resistance of S. caninervis to Hg by activating the enzymatic system of plant.

Conclusions
Our results found that Hg stress can change the ultrastructure, SS, Chl, Pro and MDA contents, SOD and POD activities of biocrust moss S. caninervis. The growth and physiological functions of S. caninervis were significantly inhibited with the increase of Hg concentration. The tolerance threshold of S. caninervis to Hg stress was 20 µM. S. caninervis can adapt to Hg stress through a series of physiological and ultrastructural changes. Changes in SOD and POD activities, Chl, SS and Pro contents were the main adaptation strategies to combat Hg stress. S. caninervis is widely distributed in Gurbantünggüt Desert, and this study indicated that it had a certain tolerance to Hg, which, until now, however, has never been reported previously. Therefore, the findings made in this paper can provide basic data and theoretical support for the environmental impact assessment of trace metals (such as Hg) on vegetation in desert area.